Characterization of a type II collagen gene (COL2A1) mutation identified in cultured chondrocytes from human hypochondrogenesis.
Characterization of a type II collagen gene (COL2A1) mutation identified in cultured chondrocytes from human hypochondrogenesis.
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DOI：
10.1073/pnas.89.10.4583
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发表时间：
1992-05
影响因子：
11.1
通讯作者：
William A. Horton;M. A. Machado;Jeffrey Ellard;D. Campbell;James Bartley;Francesco Ramirez;Emilia Vitale;Brendan Lee
中科院分区：
文献类型：
--
作者：
William A. Horton;M. A. Machado;Jeffrey Ellard;D. Campbell;James Bartley;Francesco Ramirez;Emilia Vitale;Brendan Lee
利用软骨细胞培养系统和PCR-cDNA扫描分析，在一例人类软骨发育不良的病例中检测到II型胶原基因COL2A1的微妙突变。软骨活检获得的软骨细胞在单层培养中去分化和扩大，然后通过琼脂糖培养再分化。单链构象多态性和直接测序分析发现了一个G---- a过渡，导致前α 1（II）胶原三螺旋结构域的氨基酸574位甘氨酸取代。培养过程中产生的软骨样结构的形态学评估和培养软骨细胞合成的胶原的电泳分析表明，甘氨酸取代干扰II型前胶原向胶原的转化，损害分子的细胞内运输和分泌，并破坏胶原原纤维的组装。这种实验方法对人类软骨发育不良的研究以及人类软骨细胞生物学具有广泛的意义。
A subtle mutation in the type II collagen gene COL2A1 was detected in a case of human hypochondrogenesis by using a chondrocyte culture system and PCR-cDNA scanning analysis. Chondrocytes obtained from cartilage biopsies were dedifferentiated and expanded in monolayer culture and then redifferentiated by culture over agarose. Single-strand conformation polymorphism and direct sequencing analysis identified a G----A transition, resulting in a glycine substitution at amino acid 574 of the pro alpha 1(II) collagen triple-helical domain. Morphologic assessment of cartilage-like structures produced in culture and electrophoretic analysis of collagens synthesized by the cultured chondrocytes suggested that the glycine substitution interferes with conversion of type II procollagen to collagen, impairs intracellular transport and secretion of the molecule, and disrupts collagen fibril assembly. This experimental approach has broad implications for the investigation of human chondrodysplasias as well as human chondrocyte biology.